CN114107447B - Method for quantitatively detecting novel coronavirus by combining Nb.BsrDI enzyme-mediated multiple cross-substitution amplification with fluorescence - Google Patents
Method for quantitatively detecting novel coronavirus by combining Nb.BsrDI enzyme-mediated multiple cross-substitution amplification with fluorescence Download PDFInfo
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Abstract
The present invention provides a method for detecting the binding fluorescence of a novel gene of coronavirus (SARS-CoV-2) by Nb.BsrDI (restriction endonuclease) mediated multiplex cross-displacement amplification (multiple cross displacement amplification), which is aimed at the ORF1ab and NP genes of SARS-CoV-2 to amplify and detect the nucleic acid molecules, and integrates reverse transcription, nucleic acid amplification and Nb.BsrDI mediated sequence-specific detection into one reaction, the detection result can be realized by collecting fluorescent signals, and the SARS-CoV-2 nucleic acid fragment with the minimum detectable copy number of 6.8. The method provided by the invention has the characteristics of high sensitivity and strong specificity, and can be used as a clinical and on-site detection tool.
Description
Technical Field
The invention discloses a detection method of novel coronavirus (SARS-CoV-2), belonging to the technical field of microorganism detection.
Background
The novel coronavirus pneumonia (COVID-19) is a disease caused by SARA-COV-2, the SARA-COV-2 belongs to beta coronavirus, has envelope, has round or oval particles, is often polymorphic, and has homology of more than 85% with bat SARS-like coronavirus (bat-SL-CoVZC). SARA-COV-2 spreads faster (R 0 3.28.28), and early-stage infected patients still have no symptoms or specific clinical symptoms (such as cough, fever, shortness of breath, etc.), are difficult to diagnose, and show obvious symptoms after at least 2 days or 2 weeks of exposure. The accurate and rapid identification of new patients with coronaries (especially asymptomatic infected persons) who have transmitted SARS-CoV-2 through close proximity contact is one of the major challenges in controlling the rapid transmission of SARS-CoV-2.
At present, diagnosis COVID-19 by detecting the nucleic acid of SARS-CoV-2 is an effective method. The method for SARS-CoV-2 detection mainly includes whole genome sequencing, RT-PCR, isothermal amplification method, gene editing technology, colloidal gold immune technology, etc. The whole genome sequencing has high flux and better accuracy and precision for detecting SARS-CoV-2, but the method has longer time consumption and complex operation, and is not suitable for large-scale detection; RT-PCR has higher sensitivity, specificity and accuracy for the diagnosis of COVID-19 infection, but has higher false negative detection rate (only about 47-60% of positive COVID-19 cases can be detected), and the detection time is about 2 hours; the gene editing technology has higher sensitivity and specificity, but requires special reagents and professionals; immune colloidal gold technology and enzyme linked immunosorbent technology lack specificity and sensitivity; the current primary detection method is the RT-PCR method.
Therefore, there is an urgent need to establish a detection method with high sensitivity, high specificity and high speed. The isothermal amplification technology is a tool with simple operation, short reaction time, high specificity and high sensitivity, and is beneficial to large-scale detection. The object of the present invention is to devise a novel diagnostic COVID-19 technique, termed nucleic acid endonuclease restriction mediated real-time reverse transcription multiple cross-over displacement amplification (E-rRT-MCDA). The E-rRT-MCDA method combines isothermal amplification, reverse transcription and endonuclease cleavage with real-time fluorescence analysis.
Disclosure of Invention
Based on the above objects, the present invention provides a method for amplifying a target gene by restriction enzyme-mediated multiplex cross-substitution, comprising the steps of:
(1) Extracting genome of a sample to be detected;
(2) Providing replacement primers F1 and F2; cross primers CP1 and CP2; amplification primers C1 and C2, amplification primers D1 and D2, amplification primers R1 and R2, and simultaneously linking a sequence shown as SEQ ID NO. 21 at the 5' end of the amplification primer C1, D1 or R1, wherein the sequence can be identified by restriction endonuclease Nb. BsrDI enzyme and marked by a fluorescent group, and a fluorescence quenching group is linked in the middle of the primer;
(3) Performing isothermal amplification on the genome obtained in the step (1) under the action of the primer in the step (2) and DNA polymerase to form a double-stranded amplification product, wherein in a specific embodiment of the invention, the DNA polymerase is Bst 2.0;
(4) Shearing the double-stranded amplification product obtained in the step (3) by using Nb.BsrDI enzyme;
(5) Detecting the fluorescence signal of the sheared product obtained in the step (4).
In a specific embodiment of the invention, a fluorescent quantitative PCR instrument is used for detecting the fluorescent signal of the amplified product in the step (3), a real-time turbidity gene detection system can also be used for verifying the amplification effect of the amplified product after multiple cross-displacement amplification, and a turbidity curve can be generated when the amplified product is positive, and a turbidity curve is negative and has no turbidity curve.
In a preferred embodiment, the fluorophore of step (2) is FAM, the fluorescence quenching group HBQ1, or the fluorophore is CY5, the fluorescence quenching group HBQ2. In the practice of the present invention, the above-described labeling can accomplish the detection objective, and when two different genes of interest are detected simultaneously, different fluorophores can be labeled for the amplification products in different primer combinations.
In a more preferred embodiment, the primer of step (2) is: the sequences are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2 of displacement primers ORF-F1 and ORF-F2; the sequences are respectively shown as a cross primer ORF-CP1 and an ORF-CP2 shown as SEQ ID NO. 3 and SEQ ID NO. 4; amplification primers of ORF-C1 and ORF-C2 with sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the sequences are respectively shown as an amplification primer ORF-D1 and an amplification primer ORF-D2 shown as SEQ ID NO. 7 and SEQ ID NO. 8; the sequences are respectively shown as an amplification primer ORF-R1 and an amplification primer ORF-R2 shown in SEQ ID NO. 9 and SEQ ID NO. 10. The primer combination is designed for multiplex cross-substitution amplification of novel coronavirus (SARS-CoV-2) ORF1ab (open reading frame 1 a/b), wherein, since the genome of SARS-CoV-2 is RNA, step (1) further comprises the step of reverse transcription of the genomic RNA sequence of the sample to be detected into cDNA using a reverse transcriptase.
More preferably, the amplification primer ORF-D1 of step (2) is labeled with a fluorescent group FAM.
In another preferred embodiment, the primer of step (2) is: the sequences are respectively shown as a displacement primer N-F1 and a displacement primer N-F2 shown as SEQ ID NO. 11 and SEQ ID NO. 12; the sequences are respectively shown as a cross primer N-CP1 and a cross primer N-CP2 shown as SEQ ID NO. 13 and SEQ ID NO. 14; amplification primers of N-C1 and N-C2 with sequences shown as SEQ ID NO. 15 and SEQ ID NO. 16 respectively; amplification primers N-D1 and N-D2 with sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18 respectively; the sequences are respectively shown as an amplification primer N-R1 and an amplification primer N-R2 shown in SEQ ID NO. 19 and SEQ ID NO. 20. The primer combination is designed for multiplex cross-substitution amplification of novel coronavirus (SARS-CoV-2) NP (nucleoprotein), wherein, since the genome of SARS-CoV-2 is RNA, the step (1) further comprises the step of reverse transcription of the genomic RNA sequence of the sample to be detected into cDNA by using reverse transcriptase.
In a more preferred embodiment, the amplification primer N-D1 of step (2) is labeled with a fluorescent group CY5.
More preferably, the isothermal amplification of step (3) is performed in an environment of 60-66 ℃.
Next, the present invention provides a set of primers for multiplex cross-substitution isothermal amplification of ORF1ab gene of SARS-CoV-2, said primers comprising: the sequences are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2 of displacement primers ORF-F1 and ORF-F2; the sequences are respectively shown as a cross primer ORF-CP1 and an ORF-CP2 shown as SEQ ID NO. 3 and SEQ ID NO. 4; amplification primers of ORF-C1 and ORF-C2 with sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the sequences are respectively shown as an amplification primer ORF-D1 and an amplification primer ORF-D2 shown as SEQ ID NO. 7 and SEQ ID NO. 8; the sequences are respectively shown as an amplification primer ORF-R1 and an amplification primer ORF-R2 shown as SEQ ID NO. 9 and SEQ ID NO. 10.
In a preferred embodiment, the sequence shown in SEQ ID NO. 21 is linked at the 5' end of the amplification primer ORF-D1 and is labeled with a fluorescent group, and a fluorescence quenching group is linked in the middle of the primer.
The invention also provides another set of primers for multiplex cross-over displacement isothermal amplification of the NP gene of SARS-CoV-2, said primers comprising: the sequences are respectively shown as a displacement primer N-F1 and a displacement primer N-F2 shown as SEQ ID NO. 11 and SEQ ID NO. 12; the sequences are respectively shown as a cross primer N-CP1 and a cross primer N-CP2 shown as SEQ ID NO. 13 and SEQ ID NO. 14; amplification primers of N-C1 and N-C2 with sequences shown as SEQ ID NO. 15 and SEQ ID NO. 16 respectively; amplification primers N-D1 and N-D2 with sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18 respectively; the sequences are respectively shown as an amplification primer N-R1 and an amplification primer N-R2 shown in SEQ ID NO. 19 and SEQ ID NO. 20.
In a preferred embodiment, the amplification primer N-D1 is linked at its 5' end to the sequence shown in SEQ ID NO. 21 and is labeled with a fluorescent group, and a fluorescence quenching group is linked in the middle of the primer.
Finally, the invention provides a gene detection kit containing the primer, which also comprises a chain-shifting polymerase Bst 2.0, a melting temperature regulator and a restriction endonuclease Nb.BsrDI.
The target genes of the restriction enzyme-mediated multiple displacement cross amplification detection provided by the invention are the ORF1ab sequence and the NP sequence of SARS-CoV2, and can finish detection within 65 minutes (comprising sample treatment for 10 minutes, RNA extraction for 15 minutes and detection for 36 minutes). The method has excellent detection sensitivity, the detection limit is 6.8 copies, and the method also has the advantage of detection speed, and the amplification product result can be obtained only in 36 minutes.
The specificity of SARS-CoV-2-E-rRT-MCDA technology was evaluated using pathogens (coronavirus HKU1, HIV, H9N2 influenza A, H7N9 influenza A, H5N1 influenza A, H3N2 influenza A, H1N1 influenza A, HBV, shigella, salmonella, mycobacterium tuberculosis, bacillus anthracis, klebsiella pneumoniae, pseudomonas aeruginosa, staphylococcus aureus, streptococcus pneumoniae, streptococcus suis, haemophilus influenzae, brucella, neisseria) as templates. The result shows that SARS-CoV-2-E-rRT-MCDA technology can accurately identify SARS-CoV-2, and shows that the specificity of SARS-CoV-2-E-rRT-MCDA method is good, and also proves that the primer sequence for MCDA provided by the invention has excellent specificity and amplification effect.
Drawings
FIG. 1.E-rRT-MCDA operation flow and schematic diagram;
FIG. 2. Schematic representation of the position and orientation of E-rRT-MCDA primer design;
FIG. 3.E-rRT-MCDA amplification assay result profile;
FIG. 4.E-rRT-MCDA test result map of optimal reaction temperature of ORF1ab gene;
FIG. 5 shows a graph of the test results of the optimal reaction temperature for the detection of NP gene by E-rRT-MCDA;
FIG. 6.E-rRT-MCDA shows a map of the sensitivity test results of ORF1ab gene detection;
FIG. 7.E-rRT-MCDA shows a map of the sensitivity test results of NP gene detection.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way.
The specific operational flow of the E-rRT-MCDA method of the invention is shown in FIG. 1. The procedure included sample collection, template preparation, E-rRT-MCDA reaction and detection of 4 fractions as a result.
E-rRT-MCDA amplification principle
The principle of MCDA amplification is shown in chinese patent application 201518765. X, the disclosure of which is incorporated herein by reference. The invention selects two targets of ORF1ab fragment and NP gene of SARS-CoV-2 as target sequence/target gene of MCDA amplification, and simultaneously evaluates the single amplified ORF1ab sequence or NP gene to establish a method for detecting novel coronavirus by MCDA amplification.
(1) Construction of detectable primers by MCDA amplification
The MCDA reaction system for both targets of the ORF1ab and NP genes included 10 primers (as shown in table 1) respectively, 10 regions of the recognition target sequence, 2 replacement primers, i.e., F1 and F2,2 crossover inner primers, i.e., CP1 and CP2,6 amplification primers, i.e., D1, D2, C1, C2, R1 and R2, respectively. To construct a detectable product, FAM was labeled at the 5 'end of the primer ORF-D1 amplified against the ORF1ab sequence and BHQ1 (denoted ORF-D1 x) was ligated into this primer, CY5 was labeled at the 5' end of the primer N-D1 amplified against the NP gene sequence and BHQ2 (denoted N-D1 x) was ligated into this primer, and the principle schematic is shown in fig. 1. In FIG. 1, FAM represents 6-carboxyfluorescein; CY5 represents CY5 fluorescein; BHQ1/BHQ2 represents a fluorescence quenching group; AMV stands for reverse transcriptase; bsrDI stands for restriction enzyme, recognition site TGCAATG; bst2.0 represents a chain-shifting polymerase.
(2) The detection principle of E-rRT-MCDA is briefly described below in connection with the accompanying figure 1 of the specification:
In the SARS-CoV-2-E-rRT-MCDA assay system, the short sequence (TGCAATG) recognized by the Nb.BsrDI enzyme is tagged with FAM and linked to the BHQ1 fluorescence quenching group at the 5 'end of the ORF-D1 of the ORF1ab sequence amplification primer, the short sequence (TGCAATG) recognized by the Nb.BsrDI enzyme is tagged with CY5 and linked to the BHQ2 fluorescence quenching group at the 5' end of the N-D1 of the NP gene sequence amplification primer. The RNA of a sample to be detected is subjected to reverse transcription into cDNA by reverse transcriptase (AMV), the cDNA template target gene is subjected to isothermal amplification (working temperature is 63-66 ℃) by a replacement primer, a cross primer, an amplification primer and a DNA polymerase (Bst 2.0) and a melting temperature regulator, when double chains are formed by amplification, the target gene linked with a short sequence (TGCAATG) is recognized and sheared by Nb.BsrDI enzyme, and a fluorescent group FAM/CY5 and a quenching group BHQ1/BHQ2 are broken and separated to release a fluorescent signal, and the fluorescent signal is detected by a fluorescent quantitative PCR instrument.
TABLE 1 primer sequences and modifications designed for F1ab and N genes
a ORF1a/b: open reading frames 1a/b; NP: a nucleoprotein gene.
b FAM: 6-carboxyfluorescein; CY5: CY5 fluorescein; BHQ1: a fluorescence quenching group 1; BHQ2: a fluorescence quenching group 2.
c And (2) mer: monomeric unit (monomer units); nt: nucleitide (nucleotides).
2. Reagents and instrumentation according to embodiments of the invention:
Reagents according to the examples of the present invention: reverse transcriptase (AMV) and restriction enzyme (nb.bsrdi) were purchased from beginner biotechnology limited. Isothermal amplification kit (Isothermal Amplification Kit) was purchased from Beijing-haitai-zhengyuan biotechnology limited (Beijing HaiTai-Zhenyuan co.ltd., beijin, china). RNA extraction kits were purchased from Tianlong biotechnology Co., ltd (China, western An). DNA extraction kit (QIAAMP DNA MINIKITS; qiagen, hilden, germany) was purchased from Qiagen, germany. The rest reagents are all commercially available parting pure products.
The main instrument used in the experiment of the invention: full-automatic nucleic acid extractor (GeneRotex, 96), siamion, china; fluorescent quantitative PCR instrument (7500 FAST), product of Bio-Rad, U.S.A.; real-time turbidity gene detection System (LA-500), a biological technology (China) Co., ltd.
3. Methods and bacterial strains according to embodiments of the invention
Genome extraction: plasmid DNA containing the ORF1ab sequence and NP gene sequence of the novel coronavirus, bacterial genomic DNA and other viral genomic nucleic acid were extracted using Qiagen DNA extraction kit, and the procedures were performed according to the instructions. The concentration and purity of genomic DNA were determined using a nucleic acid quantitative analyzer, and plasmid DNA was serially diluted 5-fold with GE buffer (1.1X10 5 copies, 2.1X10 4 copies, 4.3X10 3 copies, 8.5X10 2 copies, 1.7X10 2 copies, 3.4X10 1 copies, 6.8 copies, 1.4 copies). The various genome DNA are sub-packaged in small quantity and preserved at-20 ℃ for standby. The novel coronavirus sample nucleic acid is extracted by a full-automatic nucleic acid extractor and a matched RNA extraction kit (Siami Tianlong) and is operated according to the specification.
The serially diluted plasmid DNA of the novel coronavirus ORF1ab sequence and NP gene sequence is used for the exploration of the optimal temperature for MCDA amplification and the establishment of an amplification system. The specificity of SARS-CoV-2-E-rRT-MCDA technology was evaluated using common pathogen nucleic acids as templates. Bacterial strain information is shown in Table 2.
TABLE 2 viral and bacterial strain genomes used in the present invention
GZCDC: disease prevention control center in Guizhou province
4. Primer design according to the embodiment of the invention
To verify, evaluate E-rRT-MCDA technology and establish a rapid, specific and sensitive E-rRT-MCDA detection system for SARS-CoV-2. The invention designs a set of MCDA amplification primers aiming at ORF1ab sequence and NP gene sequence of SARS-CoV-2 aiming at specific genes of SARS-CoV-2, aiming at verifying feasibility, sensitivity, specificity and reliability of E-rRT-MCDA technology. The schematic of primer design is shown in FIG. 2, wherein the MCDA primer sequence of ORF1ab sequence amplifies the 13311 th to 13598 th base fragments of ORF1ab sequence of SARS-CoV-2 strain (GenBank MN 908947) genome, and the MCDA primer sequence of NP gene amplifies the 28295 th to 28547 th base fragments of N gene sequence of SARS-CoV-2 strain (GenBank MN 908947).
Example 1 feasibility of E-rRT-MCDA amplification
Standard MCDA reaction system: 2.2 μl of mixed primers including 0.4 μΜ for displacement primers F1 and F2, 2.4 μΜ for crossover primers CP1 and CP2, 1.2 μΜ for amplification primers R1, R2, D1 and D2, and 0.4 μΜ for amplification primers C1 and C2; 6mM MgSO4; 1.4mM dNTPs; 12.5. Mu.L of polymerase buffer; 1. Mu.L AMV enzyme (10U); 1. Mu.L of strand displacement Bst DNA polymerase (10U); 1. Mu.L of Nb.BsrDI enzyme (10U); 2 μl of template was supplemented with deionized water to 25 μl. The whole reaction was kept at a constant temperature of 65℃for 36 minutes.
The product after the MCDA amplification can be detected by a fluorescent quantitative PCR instrument, and positive has an amplification curve and negative has no amplification curve. The fluorescence of the MCDA primer designed for the ORF1ab gene of SARS-CoV-2 was FAM (as shown in FIG. 3A1: ① for 2.1X10 4 copies, ② for 4.3X10 3 copies, NC for negative control as H7N9 nucleic acid template, DW for blank control as sterile water). The fluorescence of the MCDA primer designed for the NP gene of SARS-CoV-2 was CY5 (see FIG. 3B1: ① for 2.1X10 4 copies, ② for 4.3X10 3 copies, NC for negative control H7N9 nucleic acid template, DW for blank control sterile water).
In addition, the invention also applies a real-time turbidity gene detection system to verify the amplification effect of the product after MCDA amplification, and positive can generate a turbidity curve and negative turbidity-free curves. Detection of the amplified product of MCDA against ORF1ab gene of SARS-CoV-2 is shown in FIG. 3A2: ① Represents 2.1X10 4 copies, ② represents 4.3X10 3 copies, NC represents negative control H7N9 nucleic acid template, and DW represents blank control sterile water. Detection of MCDA amplification product against NP gene of SARS-CoV-2 is shown in FIG. 3B2: ① Represents 2.1X10 4 copies, ② represents 4.3X10 3 copies, NC represents negative control H7N9 nucleic acid template, and DW represents blank control sterile water.
Example 2 determination of the optimal reaction temperature for the MCDA technique
Under the condition of standard reaction system, ORF1ab plasmid and NP plasmid template for SARS-CoV-2 virus and designed correspondent MCDA primer are added, its template concentration is 2.1X10 4 copies. The reaction was carried out at constant temperature (63-66 ℃), and the results were tested using a real-time turbidity gene detection system, and different dynamic profiles were obtained at different temperatures, ORF1ab sequence amplification (FIG. 4, A1 is the real-time turbidity curve, A2 is the turbidity accumulation curve) and NP gene sequence amplification (FIG. 5, B1 is the real-time turbidity curve, B2 is the turbidity accumulation curve). 63-66℃was recommended as the optimal reaction temperature for the MCDA primer. The subsequent verification in the invention selects 65 ℃ as a constant temperature condition for MCDA amplification. FIGS. 4 and 5 identify temperature dynamic graphs showing the detection of SARS-CoV-2 virus MCDA primer designed for the ORF1ab and NP gene sequences.
Example 3.E sensitivity of detection of Single target by rRT-MCDA
After standard MCDA amplification reaction is carried out by using serially diluted DNA extracted from ORF1ab sequence and NP gene plasmid of SARS-CoV-2 virus, the fluorescent quantitative PCR instrument is used for detection and display: the detection range of E-rRT-MCDA was 1.1X10 5 to 6.8 copies (FIGS. 6 and 7:a represent 1.1X10 5 copies, b represents 2.1X10 4 copies, c represents 4.3X10 3 copies, d represents 8.5X10 2 copies, E represents 1.7X10 2 copies, f represents 3.4X10 1 copies, g represents 6.8 copies, and h represents 1.4 copies). Negative results are shown when the amount of genomic template in the reaction system is reduced below 6.8 copies.
Example 4 determination of the specificity of the E-rRT-MCDA technique
The specificity of SARS-CoV-2-E-rRT-MCDA technology was evaluated using pathogens (coronavirus HKU1, HIV, H9N2 influenza A, H7N9 influenza A, H5N1 influenza A, H3N2 influenza A, H1N1 influenza A, HBV, shigella, salmonella, mycobacterium tuberculosis, bacillus anthracis, klebsiella pneumoniae, pseudomonas aeruginosa, staphylococcus aureus, streptococcus pneumoniae, streptococcus suis, haemophilus influenzae, brucella, neisseria) as templates. The result shows that SARS-CoV-2-E-rRT-MCDA technology can accurately identify SARS-CoV-2, and shows that the specificity of SARS-CoV-2-E-rRT-MCDA method is good, and also proves that the primer sequence for MCDA provided by the invention has excellent specificity and amplification effect. The pathogen nucleic acid templates useful in the present invention are shown in Table 2.
EXAMPLE 5 application of E-rRT-MCDA technology
In order to evaluate the detection effect of E-rRT-MCDA in practical application, 43 new cases of coronaries of nucleic acid preparation templates collected from Guizhou province were used, and SASR-CoV-2-E-rRT-MCDA was used to detect 43 nucleic acids, and at the same time, compared with the conventional fluorescence rRT-PCR method. The E-rRT-MCDA method detects 38 positives (38 parts of ORF1ab gene, 32 parts of NP gene) from 43 sample nucleic acid, and the conventional fluorescent rRT-PCR method detects 28 positives (28 parts of ORF1ab gene, 25 parts of NP gene), and the detection results are shown in Table 3. The E-rRT-MCDA method detects positive samples, and covers the traditional fluorescent rRT-PCR to detect positive samples, so that the number of the positive samples is the largest, and the detection time is the shortest (the detection can be completed within 65min, including sample processing for 10min, RNA extraction for 15min and detection for 36 min).
TABLE 3 specimen detection results of MCDA-LFB on patients with New coronal pneumonia in convalescence
Note that: + indicates positive; negative-for-sign
Sequence listing
<110> Guizhou disease prevention control center
<120> Method for quantitative detection of novel coronaviruses by multiple cross-substitution amplification mediated by Nb.BsrDI enzyme in combination with fluorescence
<160> 21
<170> SIPOSequenceListing 1.0
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<213> SARS-CoV-2
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<213> SARS-CoV-2
<400> 2
gaatttagca aaaccagcta ct 22
<210> 3
<211> 44
<212> DNA
<213> SARS-CoV-2
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ggagttgatc acaactacag ccataaaaac acagtctgta ccgt 44
<210> 4
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<212> DNA
<213> SARS-CoV-2
<400> 4
agtgcagccc gtcttacacc tgtagatgtc aaaagccctg 40
<210> 5
<211> 24
<212> DNA
<213> SARS-CoV-2
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ggagttgatc acaactacag ccat 24
<210> 6
<211> 20
<212> DNA
<213> SARS-CoV-2
<400> 6
agtgcagccc gtcttacacc 20
<210> 7
<211> 25
<212> DNA
<213> SARS-CoV-2
<400> 7
tgcaatgcct ttccacatac cgcag 25
<210> 8
<211> 19
<212> DNA
<213> SARS-CoV-2
<400> 8
cactagtact gatgtcgta 19
<210> 9
<211> 17
<212> DNA
<213> SARS-CoV-2
<400> 9
actgaagcat gggttcg 17
<210> 10
<211> 19
<212> DNA
<213> SARS-CoV-2
<400> 10
cagctgatgc acaatcgtt 19
<210> 11
<211> 19
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<213> SARS-CoV-2
<400> 11
ccccgcatta cgtttggtg 19
<210> 12
<211> 20
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agccaatttg gtcatctgga 20
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cgttgttttg atcgcgcccc gaccctcaga ttcaactggc 40
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accgctctca ctcaacatgg ctggtgttaa ttggaacgcc t 41
<210> 15
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<213> SARS-CoV-2
<400> 15
cgttgttttg atcgcgcccc 20
<210> 16
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<400> 16
accgctctca ctcaacatgg c 21
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<400> 17
tgcaatgtgc gttctccatt ctggttact 29
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<400> 18
aaggaagacc ttaaattccc tcga 24
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<400> 19
tgggtaaacc ttggggcc 18
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<211> 20
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tgcaatg 7
Claims (7)
1. A method for amplifying a gene of interest based on restriction enzyme-mediated multiple cross-substitution for non-diagnostic purposes, comprising the steps of:
(1) Extracting genome of a sample to be detected, and reversely transcribing a genome RNA sequence of the sample to be detected into cDNA by using reverse transcriptase;
(2) Providing displacement primers ORF-F1 and ORF-F2 with sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2 respectively; the sequences are respectively shown as a cross primer ORF-CP1 and an ORF-CP2 shown as SEQ ID NO. 3 and SEQ ID NO. 4; amplification primers of ORF-C1 and ORF-C2 with sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the sequences are respectively shown as an amplification primer ORF-D1 and an amplification primer ORF-D2 shown as SEQ ID NO. 7 and SEQ ID NO. 8; the sequence is shown as an amplification primer ORF-R1 and an amplification primer ORF-R2 shown as SEQ ID NO. 9 and SEQ ID NO. 10 respectively, wherein the amplification primer ORF-D1 is marked with a fluorescent group FAM, a fluorescence quenching group HBQ1 is linked in the middle of the primer, and the 5' end of the amplification primer ORF-D1 is linked with a sequence shown as SEQ ID NO. 21; and, providing replacement primers N-F1 and N-F2 with sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12, respectively; the sequences are respectively shown as a cross primer N-CP1 and a cross primer N-CP2 shown as SEQ ID NO. 13 and SEQ ID NO. 14; amplification primers of N-C1 and N-C2 with sequences shown as SEQ ID NO. 15 and SEQ ID NO. 16 respectively; amplification primers N-D1 and N-D2 with sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18 respectively; the sequence is shown as an amplification primer N-R1 and an amplification primer N-R2 shown as SEQ ID NO. 19 and SEQ ID NO. 20 respectively, wherein the amplification primer N-D1 is marked with a fluorescent group CY5, a fluorescence quenching group HBQ2 is linked in the middle of the primer, and the 5' end of the amplification primer N-D1 is linked with a sequence shown as SEQ ID NO. 21;
(3) Amplifying the target genes at constant temperature under the action of the primer in the step (2) and the DNA polymerase to form double-chain amplification products;
(4) Shearing the double-stranded amplification product obtained in the step (3) by using Nb. BsrDI enzyme;
(5) Detecting the fluorescence signal of the sheared product obtained in the step (4).
2. The method of claim 1, wherein the isothermal amplification of step (3) is performed in an environment of 60-66 ℃.
3. A set of primers for multiplex cross-over substitution isothermal amplification of the ORF1ab gene of SARS-CoV-2, said primers comprising: the sequences are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2 of displacement primers ORF-F1 and ORF-F2; the sequences are respectively shown as a cross primer ORF-CP1 and an ORF-CP2 shown as SEQ ID NO. 3 and SEQ ID NO. 4; amplification primers of ORF-C1 and ORF-C2 with sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 respectively; the sequences are respectively shown as an amplification primer ORF-D1 and an amplification primer ORF-D2 shown as SEQ ID NO. 7 and SEQ ID NO. 8; the sequences are respectively shown as an amplification primer ORF-R1 and an amplification primer ORF-R2 shown as SEQ ID NO. 9 and SEQ ID NO. 10.
4. A primer according to claim 3, wherein the sequence shown in SEQ ID NO. 21 is linked to the 5' -end of the amplification primer ORF-D1 and labeled with a fluorescent group, and a fluorescence quenching group is linked to the middle of the primer.
5. A set of primers for multiplex cross-over substitution isothermal amplification of the NP gene for SARS-CoV-2, the primers comprising: the sequences are respectively shown as a displacement primer N-F1 and a displacement primer N-F2 shown as SEQ ID NO. 11 and SEQ ID NO. 12; the sequences are respectively shown as a cross primer N-CP1 and a cross primer N-CP2 shown as SEQ ID NO. 13 and SEQ ID NO. 14; amplification primers of N-C1 and N-C2 with sequences shown as SEQ ID NO. 15 and SEQ ID NO. 16 respectively; amplification primers N-D1 and N-D2 with sequences shown as SEQ ID NO. 17 and SEQ ID NO. 18 respectively; the sequences are respectively shown as an amplification primer N-R1 and an amplification primer N-R2 shown in SEQ ID NO. 19 and SEQ ID NO. 20.
6. The primer of claim 5, wherein the amplification primer N-D1 has a sequence shown in SEQ ID NO. 21 linked at its 5' end and is labeled with a fluorescent group, and wherein a fluorescence quenching group is linked in the middle of the primer.
7. A gene detection kit comprising the primer of any one of claims 3 to 6, wherein the kit further comprises a strand displacement polymerase, a melting temperature regulator, and a restriction enzyme.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105755134A (en) * | 2016-04-11 | 2016-07-13 | 中国疾病预防控制中心传染病预防控制所 | Incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and application |
| CN111518948A (en) * | 2020-04-15 | 2020-08-11 | 贵州省疾病预防控制中心 | Method for detecting SARS-CoV-2 by combining reverse transcription multi-cross displacement amplification with nano biosensing |
| CN113186341A (en) * | 2021-03-30 | 2021-07-30 | 贵州省疾病预防控制中心 | CRISPR-mediated one-step constant-temperature amplification SARS-CoV-2 detection method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755134A (en) * | 2016-04-11 | 2016-07-13 | 中国疾病预防控制中心传染病预防控制所 | Incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and application |
| CN111518948A (en) * | 2020-04-15 | 2020-08-11 | 贵州省疾病预防控制中心 | Method for detecting SARS-CoV-2 by combining reverse transcription multi-cross displacement amplification with nano biosensing |
| CN113186341A (en) * | 2021-03-30 | 2021-07-30 | 贵州省疾病预防控制中心 | CRISPR-mediated one-step constant-temperature amplification SARS-CoV-2 detection method |
Non-Patent Citations (2)
| Title |
|---|
| Yi Wang等.Multiplex,Rapid,and Sensitive Isothermal Detection of Nucleic-AcidSequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.Frontiers in Microbiology.2016,第第7卷卷第1-14页. * |
| 多交叉置换扩增技术在单增李斯特菌检测中的应用及评价;李慧等;中国人兽共患病学报;第34卷(第2期);第99-104页 * |
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